Sodium absorption in the distal nephron through the epithelial Na+ channel, ENaC, is an important regulator of extracellular fluid volume and blood pressure. Mutations that delete the cytoplasmic c-teminus of the Beta and Gamma ENaC subunits cause increase Na+ absorption and hypertension (Liddle's syndrome). Loss of function mutations associated with Liddle's syndrome increase Na+ current by increasing the number of channels in the plasma membrane. Mutation of the C-terminal sequence PPPXYXXL in each hEnaC subunit reproduced these findings. Interestingly, this sequence is similar to internalization motifs found in a number of proteins. The goal of this application is to understand the function and regulation of hENaC to provide insight into basic mechanisms of Na+ transport and blood pressure control. we propose three specific aims; 1) To investigate the mechanism(s) of increased surface expression of hENaC caused by Liddle~s mutations. We will test the hypothesis that the PPPXyXXL motif is important for the internalization of hENaC, and that mutation or deletion of this motif decreases the rate of channel internalization. We will also test the alternate hypothesis that Liddle~s mutations increase the rate ate of insertion of hENAC into the plasma membrane. 2) In preliminary results, we found that hENaC function is regulated by the second-messengers cAMP and PKC. We will test the hypothesis that these second-messengers regulate hENaC function by altering cell surface expression. We will also test the alternate possibility that they alter channel rating. 3) The C-terminus of hENaC subunits mediate their interaction with other cellular proteins. It is likely that such interactions are important in controlling surface expression and function of hENaC. In this Specific Aim we will identify proteins that interact with the C- terminal PPPXyXXL motif. We will test specific candidate proteins for interaction with hENaC. To identify new unsuspected interactions, we will also screen a kidney cDNA library to identify interacting proteins. In the second part of this Specific Aim, we will determine the functional significance of interactions by testing their affect on hENaC function and surface expression. These studies will help us learn about basic mechanisms of regulation of hENAC and Na+ absorption, and may provide new insights into molecular mechanisms of hypertension.